Arboreal camera trap reveals the frequent occurrence of a frugivore-carnivore in neotropical nutmeg trees

Arboreal and flying frugivorous animals represent primary dispersers in the Neotropics. Studies suggest a possible compensation for the loss of large species by smaller ones with expanding rampant anthropogenic pressures and declining populations of larger frugivores. However, studies on seed dispersal by frugivores vertebrates generally focus on the diurnal, terrestrial, canopy, and flying species, with the nocturnal canopy ones being less studied. Setting camera traps high in the canopy of fruiting nutmeg trees revealed for the first time the high frequency of the kinkajou (Potos flavus, Schreber, 1774, Procyonidae), an overlooked nocturnal frugivore species (Order Carnivora) in the Guianas. The diversity of the fruit species consumed by the kinkajou calls for considering it as an important seed disperser. The overlap of the size of seeds dispersed by frugivores observed in nutmeg trees suggests that the small (2–5 kg) kinkajou may compensate for the loss of large (5–10 kg) frugivorous vertebrates in the canopy. Camera traps visualise how the kinkajou is adapted to forage in the nutmeg tree crown and grab the fruit. Such information is vital for conservation because compensation of seed dispersal by small frugivores is crucial in increasing anthropogenic stressors.

www.nature.com/scientificreports/ Ecuador, and the potential existence of different species within the genus Potos 27 . However, the development of safer single-rope tree climbing 28 and remote camera trapping 29 offers new opportunities to observe and analyse their behavioural ecology in treetops. Indeed, Gregory et al. (2017) recorded that 94% of canopy activity in natural branches happened at night 30 . Nutmeg tree species (Myristicaceae) form a pantropical family that heavily relies on animals for their seed dispersal, thus are excellent ecological models for the study of animal-plant interactions 31 . In neotropical biomes, primates and toucans are the primary consumers and dispersers of nutmeg tree seeds 8,9,11,12,[32][33][34] . Facing an increase in anthropogenic pressure 27 , populations of large arboreal frugivores in tropical forests are threatened [35][36][37] , leading to the downsizing of frugivore communities 38,39 . Hence, it is expected the seed dispersal process to be altered, resulting in a decline in animal-dispersed tree species diversity 40,41 . Nevertheless, studies suggest the existence of possible compensation for the disappearance of large mammalian frugivores with replacement by smaller ones, among them kinkajous in disturbed forest habitats 8,42,43 . Although kinkajous rarely forage in fruiting nutmeg trees in a study in Central America 34,44 , they are known to consume and disperse nutmeg tree seeds in the Amazonian forests of the Guianas 45,46 . Observations in neotropical nutmeg (Virola spp.) tree species 45 showed that seed-dispersal for nutmeg trees is not disrupted when large diurnal, arboreal frugivores are missing 8 . Given that kinkajou has a body mass (2-5 kg) similar to that of the diurnal frugivores brown capuchin (Sapajus apella, Linnaeus 1758) 20 and a dietary diversity overlapping that of the black spider monkey (Ateles paniscus, Linnaeus 1758), we assume that kinkajous are significant seed dispersers 24 . Here, we hypothesised that kinkajous might compensate for the loss of other larger arboreal frugivores among plant species that share a diversified array of consumers.
To test this hypothesis, we used camera traps, which are an effective method for studying arboreal fauna 29,[47][48][49] . The cost of detecting nocturnal animals such as kinkajous is reduced by half when using camera traps instead of line transect survey 50 . In previous studies, frugivores were observed in nutmeg trees from the ground 12 . Others followed animals using arboreal camera traps studies in trees using easy and safe climbing gear 29,47,50 . We, therefore, set up camera traps directly in two well-known Virola species (height between 30 and 40 m) in the forest near the newly-constructed National 2 Road (Fig. 1). We sampled 12 trees during the fruiting peak season (December-January): Virola kwatae, Sabatier 1997 (7 trees) and V. michelii, Heckel 1898 (5 trees). Through analysis of camera trap images, this study aimed to assess the occurrence of the frugivore kinkajou in Virola trees compared to other diurnal and nocturnal counterparts. Because the varying capacities of foraging animals to disperse seeds creates selective pressures on seed size 51 , we supplement our results with a literature-review-based analysis of the average seed size dispersed by small-to medium-and large-bodied frugivores.
Based on estimates from the literature, the overall distribution of average seed width dispersed by the frugivores that visit Virola trees shows similar patterns among species and groups (Fig. 4). Dunn tests corroborate these observations and highlight trends among toucans to disperse seeds significantly smaller (median width and length = 5.77 × 8.5 mm) than other larger vertebrate species (details in Tab. S3, S4, S5, S6, S7, and Fig. S4). The arboreal vertebrate species show similar patterns in the distribution of dispersed seed dimensions (Fig. 4, S4) with similar median seed width (A. paniscus = 9 mm; P. flavus = 10 mm; A. macconnelli = 9 mm; S. apella = 8 mm) and similar median seed length (A. paniscus = 15 mm; P. flavus = 15.5 mm; A. macconnelli = 15.5 mm; S. apella = 14.5 mm).

Discussion
Kinkajou (Potos flavus) frequently visited the two species of Virola nutmeg trees during the peak fruiting season at the study site (see Tab. S2, Fig. S5). Our study shows for the first time that kinkajous are very common in nutmeg trees. At the same time, the primary consumer (i.e., black spider monkey, Ateles paniscus) is nearly absent in the study forest.
The analysis of the activity of kinkajous in trees suggests that fruiting nutmeg trees are often visited when fewer alternative fruit resources are available in the early rainy season, a period with low fruit production 55,56 . The observed occurrence of kinkajous in fruiting Virola trees maybe related to a deficit of the larger diurnal, arboreal  www.nature.com/scientificreports/ Consequently, fruit surveys on the ground had shown a more significant proportion of Virola fruit with single valve husks at disturbed vs control forests 57 . However, the difference in seed removal rates across sites suggested that secondary consumers played an important role as alternative seed dispersers (e.g., toucans), eventually compensating for the disappearance of the primary seed dispersers 57 . This study shows that the nocturnal frugivore kinkajou complements the effects of birds as seed dispersers of Virola seeds. During the first hours after sunset, kinkajou travel from its diurnal shelter to feed on Virola fruit (Fig. 3a) and return before dawn to their daily sleeping roost located further away from the feeding trees 46 . This behaviour has never been documented since studies of frugivory traditionally focus on diurnal frugivores.
The review of the size of seeds dispersed by the main frugivores observed and known to forage in nutmeg trees showed the significant overlap between frugivores. This result supports the hypothesis that smaller animal species might compensate for the absence of large ones. In particular, the pattern of seed size highlights the role of kinkajous, howler monkeys, and brown capuchins as potential replacement species for spider monkeys in human-affected forest areas. Moreover, studies have shown that kinkajous disperse seeds on average 200 m away and up to 300 m from parent trees 46 . In comparison, howler monkeys and brown capuchins disperse seeds up to 390 m and 225 m, respectively on average 58 , within more extensive home ranges than kinkajous 59,60 . On the other hand, spider monkeys seem to disperse seeds between 150 and 250 m, depending on sites 58 . Although these observations support the possible compensatory role of these three species, in the absence of spider monkeys for seed dispersal, howler monkeys and brown capuchins remain preferred hunting targets 36 . In addition, howler monkeys and brown capuchins also only shared 35 to 59% overlap in fruit feeding species with spider monkeys 61 and presented a more diversified diet composed of leaves and arthropods 59,60 at that time, making them less dependent on the availability of ripe fruits. Contrastingly, kinkajous are barely hunted and remain abundant in many forest areas 62 . As a frugivore generalist and opportunist, kinkajous can eat similar plants as black spider monkeys with a similar digestive retention time 63 in the Guianas, as observed for the Central American spider monkey in Panama 24 . Like neotropical primates, kinkajous are adapted to forage into the canopy using their prehensile tail 64 . It allows them to move on small branches 65 where they can reach and grasp fruit using their forepaws, teeth, snout or nose 66,67 . All previous facts support the hypothesis that kinkajous may play a significant role in compensating seed dispersal of nutmeg trees and, more generally, for a fraction of the fruits usually consumed by spider monkeys.  www.nature.com/scientificreports/ These observations highlight the importance of studying the role of kinkajous and other nocturnal species such as olingos (Brassaricyon gabbii, Allen 1876) in seed dispersal. Olingos are understudied as they were traditionally confused with kinkajous because of their similar appearance. However, a study in Panama suggested that olingos share some ecological and behavioural traits with kinkajous and may disperse ingested seeds 68 . Since almost no studies have focused on their potential role for seed dispersal, there is thus a clear need to increase research on this group to better understand their ecological importance, especially in disturbed forests. Similarly, little is known about other diurnal frugivorous carnivores such as the coati (Nasua nasua, Linnaeus 1766), which could also play a key role in seed dispersal in fragmented forests where it can persist 69 , as well as the tayra (Eira barbara, Linnaeus 1758) that could also be involved in supporting seed dispersal processes as it also consumes fruits 70 .
In this study, another arboreal, nocturnal frugivore was recorded once: Caluromys philander (Linnaeus 1758). This marsupial consumes 25 different fruit species in the forests of Cabassou, French Guiana, including three nutmegs species 20 . At the Piste de Saint-Élie, French-Guiana, C. philander was not eating any Virola fruits. Still, it seems to share a large part of its diet composed of flowers and small-seeded fruits during the dry and wet season, respectively, with kinkajous 45 . In addition, our seed-size review demonstrated its capability to disperse seeds slightly smaller than those of kinkajous (not significant, Tab. S4,S6, S7). Even though C. philander was only observed once here, its presence in a wide range of habitats in French-Guiana 71 without being threatened by hunting suggests that it could play an important role in dispersing small seeds 72 , as indicated for small rodents 73 .
Seed size can be a fundamental threshold level for seed dispersal, especially for birds. This review highlighted a significant trend for toucans and the Guianan Cock-of-the-rock (Rupicola rupicola, Linnaeus 1766) to consume and disperse smaller seeds. Although Cock-of-the-rock was not observed in the study forest, this smaller bird is known to disperse large seeds (Tab. S7), including species consumed by spider monkeys, toucans, and kinkajous (P-M. Forget, pers. obs). Larger birds swallow these seeds, such as the guan (Penelope marail). The gizzard of this species is only slightly muscular, allowing the seed to pass through the whole digestive tract without being   74,75 . Conclusion on toucans need to be tempered and could be explained by a possible site effect. Indeed, Mata Atlantica data were used to describe seed size consumed by toucans. However, seeds from Mata Atlantica are possibly smaller than in French Guiana, which could bias our results. This highlights the clear need for studies to evaluate their role in seed dispersal compensation. This study points out the importance of smaller vertebrates for seed dispersal and explores a new approach for studying arboreal frugivores with camera traps. Beyond the difficulty of finding the most suitable location for setting up cameras to maximise the chance of capturing animals, implementing this methodology involves very tedious work that requires time, climbing skills, specific equipment to access the upper branches in the tree crown, and the presence of at least two people capable of climbing to ensure safety 28 . During this study, approximately two trees were equipped/unequipped at a height over 30 m per day by two climbers. The number of climbs is a limitation that complicates the ability to increase the number of fruit trees and sites covering a large area, i.e., that of the home range of frugivores to be inventoried far from forest access points. In addition, even when fixed directly to the trunk or the main branches, unavoidable swinging movements of the crown can cause many triggers of non-target stimuli 49 , especially during the day when the wind blows. Saturation of the memory card follows, plus battery draining faster than expected, making it impossible to maintain traps in place for more than two months without a checkup. It also underestimates frugivorous bird frequency that perches on smaller branches away from the trunk. For instance, toucans were recorded only with 33 independent events while they are known to be a regular consumer of Virola fruit and little affected by hunting in this area 8,9,43 . Future studies should consider setting up supplementary cameras in adjacent trees to observe frugivorous birds as Zhu et al. (2021) 76 did and succeeded in recording many of their interactions with fruits.
Despite these constraints, arboreal camera traps allowed for continuous, non-invasive observation of animals in the canopy, both day and night, providing a wealth of information (diversity, activities, and behaviour) about the frugivore guild of Virola spp. in the area. Specifically, this method effectively studied nocturnal frugivores, such as kinkajous, in Virola spp. and many other staple fruit resources for arboreal mammals and birds (e.g., Tetragastris spp. 13,44 ). The effectiveness of this method is consistent with the finding of a study from Bowler et al. (2017) 47 who were also able to obtain a large number of images of kinkajous without ever observing it during counts and transect observations. Schipper et al. (2007) 77 showed that standard flash photography in camera traps leads to avoidance behaviour in kinkajou. Here, the use of red infrared moving sensor camera traps suggested by Schipper et al. (2007) 77 minimised disruption to the foraging behaviour of the animals, as evidenced by a large number of kinkajous captured. It is also the case for primates who look at the lens, sometimes touching it (S. apella), but do not linger on it and do not seem bothered by camera traps when feeding. Therefore, it favours using these devices to characterise arboreal fauna. This method is currently promising for studies in the canopy 29,40,[47][48][49]51 .
During this study, thanks to many recorded events for kinkajous (N = 280, during 1320 trap nights), we showed how arboreal camera trapping protocols in targeted fruiting trees could complement the classical radiotracking of animals foraging in their home range. Firstly, camera traps can help survey several independent populations of frugivores in various habitats and feeding trees. In contrast, radio-tracking is often restricted to fewer individuals in their respective home ranges. Secondly, one can further document interactions with other frugivores species in the canopy within-tree and across plant species during their daily or nocturnal activities. And finally, camera traps record all behavioural attitudes of animals, whether they are moving along the trunk or within the branches of the canopy, handling and consuming fruit, swallowing seeds, resting, or simply visiting the treetops. These observations are simply impossible from the ground at night.
Cameras might, therefore, successfully be implemented in addition to line transects 47 , eco-acoustics 9 , or radio-tracking 10,42 to obtain the best overview of the diversity and behavioural ecology of the arboreal and flying fauna. Camera traps revealed the high frequency of an overlooked nocturnal species for the first time, suggesting its potential role in compensating the loss of other large frugivorous vertebrates such as black spider monkeys for nutmeg trees seed dispersal. Camera traps also allowed visualising how frugivore species foraging within the canopy reach ripe fruit and how they behave, highlighting the advantage of primates, which can pick, open, and peel fruit. Kinkajous handle the dehiscent fruit, and swallow seed and aril. They may pull the twig with the open fruit, and swallow the arilled seed. Then, they later defecate cleaned seeds in their droppings, similarly to primates such as spider monkeys, capuchins and howler monkeys (Forget, P-M, pers. obs.; dual observation along with Didier Julien-Laferrière in February 1986 at Piste de Saint-Elie). Such information is vital for conservation because compensation for seed dispersal by small frugivores is crucial in the context of increasing anthropogenic disturbance.
To conclude, the extension of protocols such as this one to other fruiting trees is essential to measure the impact of human disturbance on the ecology and dynamics of tropical forests. Fruit-eating species compensation seems to be mainly dependent on the diet of the smallest frugivores, as pointed out by Boissier et al. (2020) 8 , who observed a significant impact of anthropisation on the dispersal of Sapotaceae. In contrast, it was more moderate for trees of the families Myristicaceae and Burserasae, whose fruits are mainly consumed by mammals and birds 13,78 . It is also expected to see fruiting plants with the largest seeds most affected by the loss of larger frugivores, as just a few large frugivores (A. paniscus) can consume and disperse these seeds, whereas other species such as kinkajous are too small to consume these seeds (see Fig. 4, S4). The use of camera traps in anthropised areas appears necessary to characterise the canopy fauna more completely by including all nocturnal species likely to participate in seed dispersal processes. Identifying these species is a crucial point that would allow a better understanding of the sharing of resources between diurnal and nocturnal species and a better understanding of seed dispersal compensation processes made possible by the functional redundancy of the ecosystem.

Material and methods
Study site. The study was conducted in French Guiana (Fig. 1). The climate is equatorial and characterised by turns between a wet season from December to July and a dry season from August to November. Peak fruiting period occurs between March and May 55,56 . The study site was based in an anthropised forest in Eastern French Guiana (Fig. 1). More precisely, sampled trees were located near (< 1 km) the ecological corridors implemented along the National 2 Road (RN2 in french) that facilitate animal passage on the ground or in the canopy (Fig. 1). Because the road follows the hilly topography of the forest, these corridors are at elevations ranging from 30 to 90 m.
Study tree species. Virola kwatae and V. michelii (Myristicaceae) are two dioecious trees of the canopy in the Guiana Shield with an associated frugivores community well studied in the Nouragues Nature Reserve 12,79,80 and a disturbed forest 8,43 . Virola kwatae is more likely to be found on hills and forests on steep slopes 80 , while V. michelii is more common and can be found in high densities in secondary areas 20 . Both species fruit synchronously between November and March before the forest fruiting peak between March and May 55,79,80 . Fruiting duration is ca. two months and 3-4 months in Virola michelii and V. kwatae, respectively 78,79 . They produce a dehiscent capsule-like fruit with two valves protecting a seed covered by a red lipid-rich aril exposed when the fruit reaches maturity 12,79,80 . Seeds of V. kwatae are larger (seed size average: 2.8 × 1.8 cm) than seeds of V. michelii (seed size average: 2.0 × 1.4 cm). In this study, 12 trees were sampled in five forest areas (FA) located 1000 m from the road and corridors (Fig. 1b). The first forest area (FA01) is the closest to Saint-Georges with the lowest elevation (30-50 m), and the last forest area (FA07) is the farthest and is slightly higher (70-90 m).
Camera trapping protocol. Three models of Reconyx® camera traps were used: HC600 Hyperfire, XR6 Ultrafire, and Hyperfire2. The red infrared moving sensor and the absence of a white flash during shooting do not disturb the behaviour of nocturnal animals 77 . We set the HC600 and Hyperfire2 traps to take five photographs upon movement detection, while the XR6 Ultrafire was programmed to take three pictures and one video. We activated the video function for experimental purposes to supplement the information gathered from the photographs by documenting the behaviour of the foraging animals. We placed 34 81 , data (images) were first extracted and organised using the camtrapR® package on Rstudio® Version 1.1.463. Digikam® software tagged images displaying an animal to add the identified species name to the image's metadata. In some cases, the individuals photographed did not allow for species identification, so we tagged the pictures with the common name of the animal observed (e.g., sloth, large marsupial, large rodent). Photographs that did not identify a particular animal (blurred image, only part of the organism photographed) were classified as unidentified species. Each identification was then used to determine the independent events obtained. An event is defined by recording an individual by a camera trap. It is considered independent of another at least 30 min apart, as is usually expressed in literature [51][52][53][54] . To avoid pseudo-replicates, counting events was performed by considering traps in the same tree as a single trap. If an individual of a species is recorded by two camera traps of the same tree within a 30-min interval, then only one event is counted. Only one event is counted if more than one individual is observed in the image. Because not all trees had the same number of camera traps, we analysed only the pictures collected by two traps on each tree to ensure consistent sampling effort among the 12 sites. The two traps that provided the most images were chosen to select them. To standardise the period of data recording on each tree, only images obtained between 01/12/19 starting at 18:00 and 24/01/20 until 06:00 were analysed, a period of 55 days. The overlap® package 82 was used to produce activity plots of the main frugivores. Graphs were centred either at noon or midnight depending on their activity (e.g., kinkajou is a nocturnal frugivore, so its figure is centred on midnight) to stay consistent with their behaviour.
Seed size analysis. The average length and width of seeds consumed and dispersed by neotropical frugivores were compiled from studies conducted in French Guiana. The major frugivore species were selected even though we did not observe them in this study. Almost no data existed for toucans in French Guiana. However, the species of toucans from Mata Atlantica are very similar to those from French Guiana because they are either the same species (e.g., Ramphastos vitellinus) or species with comparable sizes and body mass (e.g., Selenidera maculirostris (160 g) in Mata Atlantica vs Selenidera piperivora (150 g) the Guianas). Therefore, we used a dataset for the group Ramphastidae from Mata Atlantica 83,84 to compare seed size among frugivores visiting Virola trees. Data for Ramphastos vitellinus, Pteroglossus aracari, and Selenidera maculirostris were selected and combined to form the toucans' group. As normality and homoscedasticity were not verified (tested with Shapiro and Levene test), testing for differences in average seed size consumed by the 9 main arboreal frugivores was done using